Macrophages and pericytes have been implicated in sprouting angiogenesis, and their dysfunction is linked to diverse pathological conditions such as diabetes, chronic inflammatory disease, and tumorigenesis. Little is known about the role of macrophages in physiological and pathological angiogenesis, despite their close association with newly growing vessels. Our laboratory studies the Notch signaling pathway in the context of vascular development and angiogenesis. Recent data from our lab supports highly novel functions for Notch1 in the macrophage: facilitation of macrophage recruitment and promotion of endothelial anastomosis, the merging of two vascular sprouts to form a functional vessel. Pericyte defects are a prominent component of diabetic vascular retinopathies, and pericytes are important for tumor angiogenesis. Our recent data supports a role for Notch signaling in the crosstalk between pericytes and endothelial cells during sprouting angiogenesis. Loss of function analysis demonstrated that Notch function in pericytes is critical for capillary and vein morphogenesis. The overall objective of this proposal is to study Notch function in peri- vascular cells in order to understand how macrophages and pericytes regulate angiogenesis. Our general strategy will combine genetic mouse modeling and complementary in vitro angiogenesis assays to determine the angiogenic consequences of Notch signaling modulation in macrophages and pericytes. In Aim I, we pursue the hypothesis that Notch functions in macrophages to promote and refine sprouting angiogenesis, including facilitation of endothelial anastomosis. To explore this hypothesis we will genetically manipulate murine Notch signaling in the retinal macrophages, and determine its contribution to angiogenesis in both physiological retinal development and a model of ischemic retinopathy. We will evaluate key ligands and Notch proteins that may participate in communication between macrophages and endothelium, to clarify the macrophage pathways that function downstream of Notch to regulate angiogenesis. In Aim II, we propose in vivo and in vitro approaches to examine the hypothesis that pericytes Notch signaling functions in vein and capillary differentiation, pericyte recruitmen, establishment of pericyte/endothelial interactions, and in pericyte- dependent stabilization of nascent vessels. We will genetically manipulate Notch activity in pericytes to assess the consequences of conditional removal of either Jagged1 or Notch1, or total ablation of Notch CSL signaling, from NG2-positive pericytes. Perictyes at the leading front of angiogenic growth will be evaluated in both developing and ischemic retinas. Additionally, the mouse ovary will be used as a model to study their role in luteal angiogenesis. Using endothelial cell/pericyte co-cultures to follow vessel formation in vitro, we will further clarify the roles of Notch and Notch ligands in endothelial cells versus pericytes. These studies elucidate novel mechanisms of angiogenesis that depend on interactions between endothelial and peri-vascular cells, and may be key to the understanding and treatment of a variety of human vascular pathologies. PUBLIC HEALTH RELEVANCE: Notch signaling is fundamental to proper vascular development, macrophage function, and tumor angiogenesis. We present data strongly implicating Notch as a regulator of sprouting angiogenesis and inflammatory responses. The studies will aid us in understanding Notch function in pathological angiogenesis and inflammation and in developing strategies and therapeutics targeted to reproductive disorders, retinal vascular disorders, tumor angiogenesis, and inflammatory conditions.